Research Article

The Differential Effects of Mutant p53 Alleles on Advanced

Murine Lung Cancer
1 1 2 3
Erica L. Jackson, Kenneth P. Olive, David A. Tuveson, Roderick Bronson,
1 1 1,4
Denise Crowley, Michael Brown, and Tyler Jacks
1
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; 2Abramson Family Cancer Research
Institute, University of Pennsylvania, Philadelphia, Pennsylvania; 3Department of Pathology, Tufts University School of Medicine
and Veterinary Medicine, Boston, Massachusetts; and 4Howard Hughes Medical Institute, Chevy Chase, Maryland

Abstract Features of late-stage disease including invasion, stromal desmo-

We report a direct comparison of the differential effects of
individual p53 mutations on lung tumor growth and
progression, and the creation of a murine model of
spontaneous advanced lung adenocarcinoma that closely
recapitulates several aspects of advanced human pulmonary
adenocarcinoma. We generated compound conditional
knock-in mice with mutations in K-ras combined with one
of three p53 alleles: a contact mutant, a structural mutant, or
a null allele. p53 loss strongly promoted the progression of
K-ras–induced lung adenocarcinomas, yielding a mouse
model that is strikingly reminiscent of advanced human lung
adenocarcinoma. The influence of p53 loss on malignant
progression was observed as early as 6 weeks after tumor
initiation. Furthermore, we found that the contact mutant
p53 R270H, but not the structural mutant p53 R172H, acted in a
partially dominant-negative fashion to promote K-ras–initiated
lung adenocarcinomas. However, for both mutants, loss-
of-heterozygosity occurred uniformly in advanced tumors,
highlighting a residual tumor-suppressive function conferred
by the remaining wild-type allele of p53. Finally, a subset of
mice also developed sinonasal adenocarcinomas. In contrast
to the lung tumors, expression of the point-mutant p53 alleles
strongly promoted the development of sinonasal adenocarci-
nomas compared with simple loss-of-function, suggesting a
tissue-specific gain-of-function. (Cancer Res 2005; 65(22): 10280-8)
Introduction
Lung cancer is the leading cause of cancer deaths worldwide,
with 157,200 deaths predicted for the year 2003 in the United States
alone (1). Adenocarcinoma, a subtype of non–small-cell lung
cancer, is the single most common form of lung cancer, comprising
f40% of cases (1). Activating mutations in the K-ras proto-
oncogene are found in f30% of human non–small-cell lung
cancers (2). Recently, several mouse lung cancer models have been
created using conditionally or spontaneously activatable alleles of
oncogenic K-ras (3–6). These models have contributed significantly
to our understanding of the role of K-ras in tumor initiation and
maintenance of the tumor phenotype but do not recapitulate all
aspects of the human disease. In particular, the lung tumors in
these models resemble early-stage human lung adenocarcinoma.
Note: E.L. Jackson and K.P. Olive contributed equally to this work. T. Jacks is an
Investigator of Howard Hughes Medical Institute. K.P. Olive is a David Koch fellow.
Requests for reprints: Tyler Jacks, Center for Cancer Research, Massachusetts
Institute of Technology, Building E17-517A, 77 Massachusetts Avenue, Cambridge, MA
02141. Phone: 617-253-0263; Fax: 617-253-9863; E-mail: tjacks@mit.edu.
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-2193
plasia, and metastasis are not prominent.
Alterations in the p53 tumor suppressor gene are also common
in non–small-cell lung cancer, occurring in f50% of cases (7). As
in other cancers, the majority of these alterations are missense
mutations that result in the accumulation of high levels of mutant
p53 protein. Missense mutations in p53 occur primarily in the
DNA-binding domain but the frequency of mutation at individual
codons varies dramatically between tumor types. The most
common p53 missense mutations in human pulmonary adeno-
carcinomas occur in descending order at codons 273, 248, 249, 245,
and 158 (8). Of note, codon 175 mutations are significantly less
common in lung adenocarcinomas than in other cancers. This
discrepancy is typically explained by the fact that different codons
vary in their exposure and susceptibility to mutagens. About 90% of
lung tumors are associated with exposure to carcinogens from
tobacco smoke and the polycyclic aromatic hydrocarbons found in
tobacco smoke preferentially bind to and form adducts with several
of the p53 codons frequently mutated in lung cancer. However,
these compounds also efficiently form adducts at codon 175,
weakening this theory (9).
An alternative explanation for the variation in mutation
frequency between tumor types is a difference in the tumorigenic
potential of individual mutant p53 proteins in different tissues. p53
mutations are commonly grouped into two classes that display
different behaviors in several in vitro assays (10). Contact
mutations alter residues that directly contact DNA whereas
structural mutations alter residues that are critical for maintaining
global domain structure (11). Structural mutants are frequently
described as more potent than contact mutants in promoting
cancer. In general, point-mutant p53 confers an adverse prognosis
in patients with pulmonary adenocarcinoma but the tumorigenic-
ity of individual mutations has not been determined (12, 13).
Tumor-derived p53 mutations may have two effects beyond loss
of function. There is evidence that mutant p53 alleles can act in a
dominant-negative manner to inhibit the function of wild-type p53
through hetero-oligomerization between mutant and wild-type p53
polypeptides. In vitro studies have shown that both classes of
mutants can exert dominant-negative effects on wild-type p53
activity with the effects being stronger for structural mutants (14).
Furthermore, certain point-mutant p53 alleles have been shown to
promote tumorigenesis through gain-of-function effects that are
not dependent on wild-type p53 (15).
We recently generated two strains of conditional point-mutant
p53 mice that allow for endogenous expression of mutant p53 on
Cre-mediated recombination. The first strain carries the contact
mutation p53R270H (homologous to human p53R273H) whereas the
second strain carries the structural mutation p53R172H (homologous
to human p53R175H; ref. 16).

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 The Effects of Mutant p53 Alleles on Lung Cancer

We report here the creation of an improved murine lung cancer Tumor size measurement. Lung and tumor areas were determined
model through the generation of compound mutant animals with using Bioquant Image Analysis software in manual measurement mode.
conditional mutations in K-ras and p53. This model closely
recapitulates several aspects of advanced human pulmonary Results
adenocarcinoma not commonly seen in existing models. We have Generation of K-ras/p53 compound conditional mutant
used this model to directly compare the oncogenic properties of mice. To assess the effect of p53 mutation or loss on K-ras-induced
different p53 mutations (p53R270H, p53R172H, and a large deletion lung tumorigenesis, compound mutant mice were generated that
mutation of p53) in the context of a single tumor type initiated by a harbored the conditional activatable LSL-K-ras G12D allele (6, 18)
defined oncogenic stimulus. Our comparison of K-ras-induced lung and combinations of three different conditional p53 alleles:
tumors with mutant or null alleles of p53 clearly shows a dominant- p53 LSL.R270H, p53 LSL.R172H, and p53 Flox (refs. 16, 19; Fig. 1A). For
negative effect by the p53 R270H allele that is not conferred by the clarity, the genotypes of the compound mutant mice will be
p53 R172H allele. Furthermore, we describe a tissue-specific gain of described in this article using the following abbreviations; the
oncogenic potential conferred by both the p53 R172H and the conditional LSL-K-ras G12D allele will be referred to as K and the
p53 R270H alleles toward the development of carcinomas of the wild-type p53, p53 LSL.R270H, p53 LSL.R172H, and p53 Flox alleles will be
sinonasal mucosa. referred to as +, 270, 172, and Fl, respectively. The compound
mutant mice will be referred to collectively as K,P mice. The
Materials and Methods following K,P mice were generated: K;Fl/+, K;270/+, K;172/+, K;Fl/Fl,
Breeding schemes. LSL-K-ras G12D mice were crossed to p53 Fl/+ mice to
generate LSL.K-ras G12D;p53 Fl/+ (K;Fl/+) mice. The p53 R172H and p53 R270H
strains were each crossed to the p53 Fl strain to generate p53 R172H/Fl and
p53 R270H/Fl mice. By crossing K;Fl/+ mice with p53 R172H/Fl and p53 R270H/Fl
mice, offspring of the following genotypes were generated for use in tumor
studies: K;Fl/+, K;172/+, K;Fl/Fl, K;172/Fl, K;270/+, and K;270/Fl.
Molecular analysis of recombination efficiency. DNA was prepared
from dissected tumors. For the K-ras allele, PCR was done using the
Advantage-GC-cDNA kit from Clontech (Mountain View, CA) with primers
flanking the lox-stop-lox cassette: K5V-1 forward, 5V-GGGTAGGTGTTGGGA-
TAGCTG-3V, and K3V-3 reverse, 5V-TCCGAATTCAGTGACTACAGATGTACA-
GAG-3V. The wild-type and recombined conditional K-ras alleles yield 265- and
305-bp products, respectively. Analysis of the conditional p53 point-
mutant alleles was done using standard PCR buffer with primers flanking
the lox-stop-lox cassette: dt020200.1 forward, 5V-AGCCTGCCTAGCTTCCT-
CAGG-3V, and dt011200.3 reverse, 5V-CTTGGAGACATAGCCACACTG-3V. The
wild-type and Flox p53 alleles produce a 291-bp product and the
recombined conditional allele produces a 325-bp product. PCR analysis of
the floxed allele was done using standard PCR buffer with primers flanking
the loxP sites in exons 2 and 10: forward, 5V-CACAAAAACAGGTTAACCCAG-
3V, and reverse, 5V-GAAGACAGAAAAGGGGAGGG-3V. The recombined allele
yields a 612-bp product; no product is produced from the wild-type allele.
AdenoCre infection. Mice were infected with 5
105 plaque-forming
units (pfu) of AdenoCre virus at 6 to 8 weeks of age. AdenoCrE:CaPi
coprecipitates were prepared as described (17). Mice were anesthetized with
avertin. AdenoCre:CaPi coprecipitates were administered intranasally in
two 62.5-AL instillations.
Tissue harvesting. Mice were sacrificed by CO2 asphyxiation. The
trachea was exposed and the lungs were inflated with formalin. All tissues
were fixed in formalin overnight at room temperature and then placed into
70% ethanol and sent for processing through paraffin. Once processed,
each lobe of the lung was cut in a set pattern and embedded in paraffin.
Remaining organs were embedded according to standard protocols.
Immunohistochemistry and trichrome staining. All lungs were
sectioned as follows: five-step sections were taken at 100 Am apart, with Figure 1. Generation of K,P compound mutant mice. A, diagram of K- ras
five unstained sections taken after section 3. p53 immunohistochemistry was and p53 conditional alleles. The LSL-K-ras G12D allele contains an activating
done following Trilogy dewaxing/unmasking according to instructions of the mutation at codon 12 and a Lox-Stop-Lox (LSL) cassette inserted into intron
0 upstream of the transcriptional start site. The p53 LSL.R172H and p53 LSL.R270H
manufacturer. p53 CM5 rabbit polyclonal antibody (Novo Castra, Newcastle
alleles contain an Arg!His mutation in exons 5 and 8, respectively, and a
upon Tyne, United Kingdom) was used at a 1:500 dilution. Staining was LSL cassette in intron 1. The p53 Flox allele encodes the wild-type p53 protein
completed with a horseradish peroxidase anti-rabbit kit (Vector Labs). For and contains LoxP sites flanking exons 2 and 10. B, recombination efficiency of
phospho-mitogen-activated protein kinase (MAPK) immunohistochemistry, conditional alleles. Genomic DNA was isolated from 10 different K;270/Fl tumors
(lanes 1-10 ). Top, to assess recombination of the LSL-K-ras G12D allele, PCR
high-temperature citrate buffer unmasking was done and anti–phospho-p42/ was done using primers flanking the LSL cassette. The recombined K-ras G12D
44MAPK (Cell Signaling, Beverly, MA) was diluted 1:100. Sections were and wild-type alleles yield 305-bp (top arrow ) and 265-bp (bottom arrow )
incubated in primary antibody overnight at 4jC. Trichrome staining was done products, respectively. Middle, PCR analysis of the p53 locus using primers
using a Masson’s trichrome kit from Poly Scientific R&D (Bayshore, NY). flanking the LSL cassette in intron 1. The 335-bp product (arrow ) is produced
from the recombined conditional allele. Asterisk, unrecombined sample. Bottom,
Tumor grading. Tumor grading was done without knowledge of genotype PCR analysis of the p53Fl allele using primers flanking exons 2 and 10. The
on the level 3 section of each lung. Each tumor was given a score of 1 to 5 recombined p53Fl allele produces a 612-bp product. No product is produced
based on predetermined criteria (see Results for specific grading criteria). from the unrecombined allele (asterisks ).

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Cancer Research
K;270/Fl, and K;172/Fl, allowing us to examine the effects of point-
mutant p53 proteins in the presence and absence of wild-type
p53 compared with the complete loss of p53. Crosses were
designed to minimize effects of mixed genetic backgrounds (see
Materials and Methods). Cohorts of K,P mice were infected
with 5
105 pfu of a recombinant adenovirus expressing Cre
recombinase (AdenoCre) by intranasal instillation and then
sacrificed at various time points (described below). As expected,
all of the mice developed multiple primary lung tumors following
infection with AdenoCre.
Infection with AdenoCre induces recombination of the
conditional alleles. To assess the recombination efficiency of each
allele when multiple alleles were present, DNA was isolated from
tumors dissected from the lungs of K;270/Fl mice and PCR was done
to detect recombination. The p53 R270H and the p53 R172H alleles only
differ at the single base mutations downstream of the LSL cassette,
so the recombination of these alleles was assumed to be identical. In
every tumor, PCR amplification of the K-ras allele produced a
product 40 bp larger than the wild-type allele due to the single loxP
site remaining after Cre-mediated recombination (Fig. 1B, top).
Therefore, the K-ras G12D allele recombined with 100% efficiency.
Similar analyses showed that the p53 LSL.R270H allele recombined in
90% (9 of 10) of the tumors analyzed (Fig. 1B, middle). The
recombination efficiency of the p53 Flox allele was comparable,
occurring in 80% (8 of 10) of tumors analyzed (Fig. 1B, bottom).
Loss of p53 function promotes malignant progression of
lung tumors. To investigate the effects of p53 loss, we evaluated
the lung tumor phenotypes of K;Fl/+ and K;Fl/Fl mice at various
time points after AdenoCre infection. A cohort of K;Fl/+ mice were
euthanized at a late time point (26 weeks) after infection. The
tumors present in K;Fl/+ mice resembled those seen previously in
L-K-ras G12D single mutant mice, ranging from adenomas to early-
stage adenocarcinomas (6). This indicates that the loss of a single
copy of p53 does not significantly affect the progression of K-ras-
initiated lung adenocarcinomas.
A cohort of K;Fl/Fl mice was also generated for analysis at this
time point. However, it became apparent that these mice would not
survive for 26 weeks after infection, so they were sacrificed after 19
weeks. In contrast to the K;Fl/+ mice, all of the K;Fl/Fl animals had
advanced pulmonary adenocarcinomas. Multiple tumors were
present in each mouse ranging from well-differentiated tumors
to advanced highly dysplastic lesions containing large sheets of
dysplastic cells, abnormal mitoses, and multinucleate giant cells.
To quantify the extent of progression of K;Fl/Fl tumors, we
devised a grading system by which to evaluate the stage of every
tumor in each mouse. Tumors were scored in a blinded manner on
a scale of 1 to 5 with grade 5 indicating the most advanced tumor
phenotype. The criteria for each grade are as follows: Grade 1
tumors have uniform nuclei showing no nuclear atypia. Grade 2
tumors contain cells with uniform but slightly enlarged nuclei
that exhibit prominent nucleoli. Grade 3 tumors have cells with
enlarged, pleomorphic nuclei showing prominent nucleoli and
nuclear molding. Grade 4 tumor cells have very large, pleomorphic
nuclei exhibiting a high degree of nuclear atypia, including
abnormal mitoses and hyperchromatism, and contain multinucle-
ate giant cells. Grade 5 tumors have all the features of grade 4
tumors and also show stromal desmoplasia surrounding nests of
tumor cells (see Fig. 2 for examples).
Using this grading scheme to analyze 944 tumors from 12 K;Fl/+
mice and 1161 tumors from 15 K;Fl/Fl mice, we confirmed that loss
of p53 resulted in a markedly more severe tumor phenotype in K-ras-
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initiated lung adenocarcinomas (Fig. 3A). In addition, K;Fl/Fl tumors
showed several characteristics of human lung tumors that were
lacking in previous murine lung cancer models (20). Perhaps, most
striking was the presence of tumors containing a large stromal
component in which nests of tumor cells could be found growing
within a field of desmoplastic stroma (Fig. 3B). Masson’s trichrome
staining of these tumors showed the abundant production of
collagen by the stromal fibroblasts (Fig. 3C) and immunohisto-
chemistry confirmed the expression of smooth muscle actin by some
of the stromal fibroblasts (data not shown).
A subset of K;Fl/Fl tumors was highly invasive, growing into the
hilus, heart, and overlying pleura (Fig. 3D). Furthermore, tumor
cells were observed along the luminal surfaces of blood and
lymphatic vessels within the tumor mass (data not shown) showing
extravasation of the tumor cells, which is required for metastatic
spread. In addition, lymph node metastases were present in over
50% (8 of 15) of K;Fl/Fl mice (Fig. 3E) and a small percentage had
metastases to distant organs (Fig. 3F).
Having established that p53 loss accelerates the progression of
K-ras-initiated lung adenocarcinomas, we were curious how rapidly
this contributed to tumor progression. Therefore, we examined the
lungs of K;Fl/+ and K;Fl/Fl mice 6 weeks after infection with
AdenoCre. The lungs of K;Fl/+ mice contained lesions ranging from
atypical adenomatous hyperplasia to small adenomas (Fig. 4A). The
adenomas present in these mice were very small and, with rare
exceptions, had regular nuclei (Fig. 4B). Atypical adenomatous
hyperplasia and adenomas were also present in K;Fl/Fl mice but
were often larger and frequently displayed nuclear atypia (Fig. 4C).
Even some of the smallest lesions had aberrant nuclei (Fig. 4D) and
a few tumors even contained multinucleate giant cells (Fig. 4C).
Grading of 111 tumors from 12 K;Fl/+ mice and 215 tumors from
K;Fl/Fl mice showed a clear shift (P < 0.001) towards more
malignant lesions in K;Fl/Fl mice (Fig. 4A), indicating that p53 loss
promotes rapid lung tumor progression.
A partial dominant-negative effect by the p53 R270H allele
towards malignant progression. Endogenous expression of
mutant p53 can result in a gain-of-function effect on tumor
development in some tissues (16, 21). Therefore, we examined
whether endogenous expression of point-mutant p53 in K-ras-
initiated lung adenocarcinomas would result in a more severe tumor
phenotype than homozygous deletion of p53. Cohorts of K;270/Fl
and K;172/Fl mice were sacrificed 19 weeks after AdenoCre infection
and their lung tumors were compared with K;Fl/Fl mice described
above. Surprisingly, grading of 841 tumors from 11 K;270/Fl mice and
839 tumors from 10 K;172/Fl mice did not reveal any differences in
the distribution of tumor grades compared with K;Fl/Fl mice
(Fig. 5A). All three genotypes had substantially fewer low-grade and
more high-grade tumors than K;Fl/+ animals sacrificed at 26 weeks
postinfection, and all had tumors that were highly invasive and often
metastatic to the regional lymph nodes. Together, these data support
the loss of wild-type p53 function as a factor in the progression of
K-ras-initiated lung adenocarcinomas but do not support a gain-of-
function effect by point-mutant p53 in these tumors.
The absence of a gain-of-function effect allowed us to cleanly
evaluate the role of dominant-negative effects of mutant p53 in the
progression of K-ras-initiated lung adenocarcinomas. Cohorts of
K;270/+ and K;172/+ mice were sacrificed 26 weeks after AdenoCre
infection and compared with K;Fl/+ mice described above. Grading
was done on 932 tumors from 11 K;270/+ mice and 773 tumors
from 10 K;172/+ mice. Strikingly, whereas the distribution of tumor
grades was similar between K;Fl/+ and K;172/+ mice, the K;270/+
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 The Effects of Mutant p53 Alleles on Lung Cancer

Figure 2. Tumor grades in K,P compound

conditional mutant mice. A, a region of
advanced adenomatous hyperplasia,
adjacent to normal lung (left ). Hyperplastic
cells are enlarged and may accumulate
slightly but generally follow the underlying
lung architecture. B, grade 1 lesions form a
solid tumor but have regular nuclei. C,
grade 2 lesions may have slightly irregular
nuclei and prominent nucleoli. D, cells in
grade 3 lesions exhibit pleomorphic nuclei,
prominent nucleoli, and nuclear molding.
E, grade 4 lesions have enlarged,
pleomorphic nuclei (single arrows ),
aberrant mitoses (double arrows ), and
tumor giant cells (triple arrows ). F, grade 5
lesions exhibit all the criteria of grade 4
lesions as well as nests of tumors cells
surrounded by a desmoplastic stroma.

mice displayed a significant shift in the tumor distribution (m2, P <
0.001) with fewer low-grade and more high-grade tumors (Fig. 5A
and B). Therefore, p53 R270H, but not p53 R172H, acted as a partial
dominant-negative allele in the development of high-grade lung
adenocarcinoma.
To determine whether this effect also extended to the size of the
tumors, we examined the tumor burden after 26 weeks of growth.
The average number of tumors in K;Fl/+, K;172/+, and K;270/+ mice
were similar (79 F 21, 77 F 24, and 85 F 17, respectively). We
measured tumor burden as the percentage of total lung occupied
by tumor. Like the grading results, we found that K;Fl/+ and K;172/+
mice had similar tumor burdens with tumor occupying 24% and 27%
of the total lung area, respectively. However, the tumor burden in
K;270/+ mice was consistently higher, with tumor comprising 36% of
the total lung area (P = 0.024; Fig. 5C). These findings further support
a dominant-negative effect specifically conferred by the p53 R270H
allele on lung tumor growth and progression.
Up-regulation of the Raf/mitogen-activated protein kinase
pathway occurs in advanced tumors. Studies in primary
fibroblasts have linked high levels of Raf/MAPK activity to Ras-
induced premature senescence, in part through activation of
p19ARF, a positive regulator of p53 (22). Therefore, we reasoned
that mutation of p53 might allow for up-regulation of this signaling
pathway, which could account for its tumor-promoting effects. To
address this question, immunohistochemistry was done on both
early- and late-stage tumors from the K,P mice of all six genotypes
using anti–phospho-p42/44MAPK antibodies.
At 6 weeks postinfection, no phospho-MAPK positive tumors
were present in any of the animals evaluated, indicating that loss of
p53 alone is not sufficient to allow up-regulation of the Raf/MAPK
pathway. However, a subset of late-stage tumors contained large
foci of phospho-MAPK positive cells and sometimes the entire
tumors were immunoreactive. The percentage of phospho-MAPK
positive tumors varied depending on the p53 genotype (Fig. 5D).
Roughly half of the tumors in K;Fl/Fl, K;172/Fl, and K;270/Fl
animals stained positively for phospho-MAPK (52.2%, 51.2%, and
50.6% respectively). Strikingly, the frequency of MAPK activation
was identical in K;270/+ mice, occurring in 53.1% of the tumors,
whereas only 13.7% of K;Fl/+ tumors were phospho-MAPK positive.
Therefore, although p53 loss alone is not sufficient to enable up-
regulation of the Raf/MAPK pathway, it may promote the
accumulation of additional events that allow MAPK activation.
Furthermore, the p53 R270H allele dominantly interferes with the
ability of wild-type p53 to restrain MAPK up-regulation.

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Wild type–specific loss of heterozygosity in K;270/+ tumors
despite dominant-negative effects by the p53 R270H allele. To
determine whether the dominant-negative effect of the p53 R270H
allele alleviates the selective pressure to lose the wild-type allele of
p53, we examined the frequency of loss of heterozygosity (LOH).
Quantitative PCR analysis was done on genomic DNA isolated from
tumors microdissected from the lungs of K;Fl/+ and K;270/+ mice
using TaqMan MGB probes. For both genotypes, 100% of the
tumors analyzed (n = 11 for K;270/+ and n = 8 for K;Fl/+) showed
loss of the wild-type p53 allele. These data show that there is still
selective pressure for full loss of p53 function despite the
dominant-negative effects of the p53 R270H allele. This may explain
why the tumors found in K;270/+ mice, although larger and more
advanced than those in K;Fl/+ mice, were not as large and
advanced as those in K;Fl/Fl mice, and may help to explain the
wild type–specific p53 LOH that is frequently observed in human
tumors (12, 13, 23).
Mutant p53 promotes the development of sinonasal
adenocarcinomas. The method of AdenoCre administration used
here exposes the entire respiratory tract to the virus. Thus,
infection and subsequent recombination of the conditional alleles
could occur in cells of the nasal passages or the trachea. However,
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none of the conditional K-ras G12D single mutant mice developed
tumors in the upper respiratory tract, suggesting that the
expression of K-ras G12D in these cells is not sufficient to induce
tumorigenesis. In the course of performing the experiments
described above, we observed that K;172/Fl and K;270/Fl
compound mutant mice had a higher mortality than K;Fl/Fl
compound mutants, which could not be explained by differences in
their lung tumor phenotype, as there was none. Histologic analysis
of head sections revealed the presence of anaplastic adenocarci-
nomas in the nasal passages of a subset of the 19-week K;Fl/Fl,
K;270/Fl, and K;172/Fl mice that seemed to arise from the mucous
glands of the nasal sinuses. The tumors were extremely poorly
differentiated but pan-cytokeratin immunostaining confirmed
their epithelial origin (data not shown). The tumor cells displayed
dramatically pleomorphic, vessiculated nuclei with prominent
nucleoli (Fig. 6B). Furthermore, the tumors were destructively
invasive, often penetrating through the cribriform plate into the
olfactory bulb (Fig. 6C).
Consistent with their increased mortality, the incidence of
sinonasal adenocarcinomas was >4-fold higher in K;172/Fl (64%)
and K;270/Fl (69%) mice than in p53Fl/Fl (14%) mice (Fig. 6D). The
increased incidence of tumors in K;270/Fl and K;172/Fl mice was
Figure 3. Loss of p53 results in
progression of K-ras -induced lung
adenocarcinomas. Slides are stained
with H&E unless otherwise indicated.
A, distribution of tumor grades in K;Fl/+
mice (blue columns ) and K;Fl/Fl mice
(red columns ). Columns, percent of total
tumors for each grad; bars, SE. B, a region
of stromal desmoplasia with invasion of
tumor cells in a grade 5 tumor;
200.
C, Masson’s trichrome stained section
of a tumor with tumors cells (red ) invading
a region of collagen-containing stroma
(blue );
400. D, section of a lung
adenocarcinoma (T ) invading adjacent
cardiac muscle (C);
200. E, section of a
lung adenocarcinoma metastasis (M ) in a
thoracic lymph node;
100. F, section of a
distal lung tumor metastasis (M ) to the
kidney. Arrows, normal glomeruli in the
kidney; dashed line, boundary of the
metastasis.
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Figure 4. Effects of p53 loss are apparent
six weeks after AdenoCre infection. Slides
are stained with H&E. A, distribution of
tumor grades in K;Fl/+ (blue columns )
and K;Fl/Fl mice (red columns ). Columns,
percent of total tumors for each grade;
bars, SE. B, section of a lesion from a
K;Fl/+ mouse, 6 weeks postinfection with
AdenoCre. Nuclei are regular and retain
apical/basal polarity;
400. C, section of
a lesion from a K;Fl/Fl mouse, 6 weeks
postinfection with 5
105 pfu of AdenoCre.
Arrows, several enlarged cells with
aberrant nuclei;
400. D, an example of
a very small lesion from a K;Fl/Fl mouse,
6 weeks postinfection with 5
105 pfu
of AdenoCre. Tumor cells have irregular
nuclei, including one enlarged, aberrant
cell (arrow );
400.
highly statistically significant (m2, P = 0.001). Given the significantly
higher incidence of sinonasal carcinoma on expression of point-
mutant p53 compared with loss of p53 expression, these data
provide strong evidence for a gain of oncogenic potential by point-
mutant p53 in vivo.
Discussion
In this work, we present compound mutant mice incorporating
somatically manipulatable conditional mutations in the K-ras
proto-oncogene and the p53 tumor suppresser gene to allow for
analysis of mutant cell behavior in the context of an otherwise
normal animal. Cooperation between oncogenic ras and point-
mutant p53 was one of the earliest examples of genetic interaction
between two cancer genes (24). Based on the observation that both
K-ras and p53 are frequently mutated in human tumors, we expect
that the K,P compound mutant mice described in this work will be
useful in accurately modeling a number of different types of cancer
beyond the advanced pulmonary adenocarcinoma described here.
K,P mice develop advanced lung adenocarcinoma. Through
the generation of K-ras G12D;p53 compound conditional mice, we
have created an improved murine lung adenocarcinoma model
that faithfully recapitulates several characteristics of the human
disease not commonly observed in previous models. The tumors in
these mice exhibit a high degree of nuclear atypia, elicit stromal
desmoplasia, and are invasive and metastatic. Although it has been
shown in other mouse lung cancer models that K-ras and p53
cooperate in the promotion of tumor growth and progression, the
induction of a desmoplastic response and the development of
metastatic disease were not reported (4, 5).
Human pulmonary adenocarcinomas often contain a large
stromal component. The microenvironment in which a tumor
resides may play a critical role in tumor growth and progression, and
interactions between cells in the tumor and the microenvironment
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The Effects of Mutant p53 Alleles on Lung Cancer
have been shown to elicit changes in cell behavior (25, 26). The
induction of desmoplastic stroma is commonly seen in non–small-
cell lung cancer and may contribute to disease progression. The
production by stromal fibroblasts of hyaluronan and various matrix
metalloproteinases involved in reorganization of the extracellular
matrix are thought to influence the invasive and metastatic
properties of the tumor (25). Correlative studies suggest that stromal
productions of the cell adhesion molecule hyaluronan and of
thymidine phosphorylase, which has angiogenic activity, correlate
with poor prognosis (27, 28).
Human non–small-cell lung cancer is highly metastatic. Patients
diagnosed with early-stage disease that has not spread to lymph
nodes or distant organs have an average 5-year survival of about
50%. However, only 15% of lung cancer patients are diagnosed at
this stage; most are diagnosed with metastatic disease and have an
overall 5-year survival rate only 14%. The current therapeutic
regimens are not effective in the treatment of advanced non–
small-cell lung cancer. Many murine lung cancer models have been
created that provide valuable insights into the molecular
alterations driving lung tumorigenesis. However, because these
models do not generally develop advanced disease, their growth
properties and response to therapeutics may not accurately reflect
those of human tumors. Thus, the model described here will
provide a valuable system for the study of lung tumor biology and
the preclinical testing of novel therapeutics.
The contact mutation p53R270H contributes more strongly to
lung tumorigenesis than the structural mutation p53R172H.
Although it is generally accepted that p53 mutation is an important
event in the development of lung cancer, the relative contribution
of different p53 mutations has not been previously analyzed in an
in vivo setting. By comparing the effects of different p53 alleles
under identical conditions, we have established that loss of p53
function is sufficient to promote the progression of K-ras initiated
lung adenocarcinomas in mice and that the p53 R270H allele more
10285 Cancer Res 2005; 65: (22). November 15, 2005
Cancer Research
strongly contributes to lung tumorigenesis by acting as a partial
dominant-negative. The primary functions of p53 are to respond to
cellular stresses by transactivating genes involved in cell cycle
arrest, apoptosis, and genome maintenance. Although we could not
determine the contribution of specific p53 effector pathways, it is
unlikely that the apoptotic functions of p53 are significant in this
setting because we could not detect any differences in the rates of
apoptosis within lung tumors of K,P mice by immunostaining for
cleaved caspase-3 (data not shown).
Instead, it seems likely that loss of the genome maintenance and
antiproliferative functions of p53 are critical to the development of
advanced lung adenocarcinoma. One of the most prominent
features of the tumors in K;Fl/Fl mice is the development of severe
nuclear dysplasia. The observation of lesions with prominent
nuclear atypia just 6 weeks after infection with AdenoCre indicates
a rapid accumulation of genetic abnormalities following loss of
p53. Our finding that early-stage K;Fl/Fl, K;270/Fl, and K;172/Fl
tumors did not up-regulate the MAPK pathway whereas late-stage
tumors did is consistent with this hypothesis. The data show that
p53 mutation is not sufficient for up-regulation of this pathway but
facilitates the acquisition of additional events leading to increased
signaling through the Raf/MAPK pathway.
Loss of p53 seems to affect the progression of lung adenocarci-
nomas rather than their initiation. Lung tumors are extremely rare
in p53/ mice, presumably due to the early onset of lymphomas
and sarcomas. A small subset of p53+/ mice does develop lung
adenocarcinomas but the tumors are generally solitary and have a
long latency (>18 months). Likewise, p53Fl/Fl mice treated with
AdenoCre also develop lung adenocarcinomas but only 1 to 1.5
years after treatment (29). Therefore, p53 mutation alone is not
sufficient for the initiation of lung cancer. Instead, activation of
K-ras serves to initiate the tumor and p53 loss contributes to the
progression.
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Research.
Although p53 loss of function is sufficient to drive tumor
progression in K-ras-induced lung adenocarcinomas, p53 is rarely
deleted in human lung tumors. Rather, p53 is commonly point-
mutated at specific hotspot residues in the DNA-binding domain in
lung and other tumors. In particular, contact mutations at codons
248 and 273 are very common in human lung adenocarcinomas. In
contrast, an analysis of spontaneous tumors from the IARC p53
mutation database (version R9) confirms that codon 175 mutations
are substantially underrepresented in adenocarcinomas and non–
small-cell lung cancers compared with all tumors (m2, P = 0.000026).
Previously, explanations for this variation focused on the mutagenic
effects of cigarette smoke on different p53 codons, especially in light
of in vitro evidence showing that codon 175 mutations are more
potent in oncogenic assays. However, we found that the p53R270H
mutation is more strongly dominant-negative over wild-type p53
than is the p53R172H mutation and therefore more effective in tumor
promotion as shown by the increased tumor burden and higher
proportion of high-grade tumors in K;270/+ mice.
There are many possible explanations for this effect. For
example, p53R270H may have a stronger intrinsic capacity to
interfere with wild-type p53 than p53R172H. Alternatively, p53R270H
may accumulate to higher levels in tumor cells than p53R172H.
Consistently, we found that a higher proportion of K; 270/+ tumors
showed strong immunoreactivity for p53 (data not shown). One
final possibility is that K;270/+ tumors undergo LOH at an earlier
point than K;172/+ tumors, perhaps due to a specific dominant-
negative effect by p53R270H on the genome maintenance functions
of p53. Once again, our data on tumor grade and MAPK up-
regulation are consistent with this hypothesis as discussed above.
Gain-of-function effects of point-mutant p53 in sinonasal
adenocarcinoma. In addition to non–small-cell lung cancer, some
of the K,P mice developed anaplastic adenocarcinoma of the nasal
mucosa arising from the mucous gland epithelium. A significantly
Figure 5. Dominant-negative effects
by the contact mutant p53R270H. A,
distribution of tumor grades in K;Fl/Fl
mice (blue columns ), K;172/Fl mice
(green columns ), and K;270/Fl mice
(red columns ). Columns, percent of total
tumors for each grade; bars, SE.
B, distribution of tumor grades in K;Fl/+
mice (blue columns ), K;172/+ mice
(green columns ), and K;270/+ mice
(red columns ). Columns, percent of total
tumors for each grade; bars, SE. C, areas
of tumors in K;Fl/+ mice (blue columns),
K;172/+ mice (green columns ), and
K;270/+ mice (red columns ). Columns,
percent of lung area occupied by tumors;
bars, SE. D, MAPK activation in K,P mice.
Columns, percent of tumors with positive
staining for $-MAPK in the indicated
genotypes.
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Figure 6. Sinonasal tumors in K,P mutant
mice. Slides are stained with H&E. A, a
small sinonasal adenocarcinoma is shown
in the nasal mucosa;
100. B, section of a
sinonasal adenocarcinoma from a K;270/Fl
mouse. These tumors develop dramatically
pleomorphic nuclear atypia;
400. C,
section of a sinonasal adenocarcinoma
invading the brain. Dashed line, location of
the cribriform plate. Arrow, leading front of
the tumor invading into the remains of the
olfactory bulb (H );
40. D, fractions of
K;Fl/Fl, K;172/Fl, and K;270/Fl mice that
developed sinonasal adenocarcinoma.
higher proportion of both K;270/Fl and K;172/Fl mice developed
sinonasal adenocarcinomas (69% and 64%, respectively) compared
with K;Fl/Fl mice (14%). Because the lung tumor phenotype did not
vary between K;Fl/Fl and K;270/Fl or K;172/Fl, the discrepancy in
sinonasal adenocarcinoma incidence strongly suggests a tissue-
specific gain-of-function of point-mutant p53. Also intriguing is the
fact that the relative effects of the p53 R270H and the p53 R172H alleles
are similar in the mucous gland epithelium but appreciably different
in the lung epithelium, also pointing to the tissue specificity of the
effects of individual p53 mutations. Given the prevalence of p53
mutations in human cancer, elucidating the different allele- and
tissue-specific effects of mutant p53 is critical to developing a more
complete understanding of molecular carcinogenesis.
Sinonasal adenocarcinomas are rare in humans and the tumors
seen in our mice are not readily diagnosed based on human
classification criteria. However, we believe they arise from the
epithelium of the mucous glands and, thus, are most similar to
minor salivary gland tumors in humans. The majority of over 500
minor salivary glands in the head and neck are mucous glands
(http://www.bcm.edu/oto/grand/7992.html). Minor salivary gland
tumors in humans account for only 10% to 15% of all salivary gland
tumors but, unlike tumors of the major salivary glands, which are
mostly benign, z80% of minor salivary gland tumors are malignant
(30, 31). Interestingly, although p53 alterations are rare when
evaluated in salivary gland carcinomas as a whole, their frequency
may be much higher in carcinomas of the minor salivary glands. An
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Acknowledgments
Received 6/22/2005; revised 9/9/2005; accepted 9/20/2005.
Grant support: Howard Hughes Medical Institute (Physician Postdoctoral
Research Fellowship) and American Association for Cancer Research-Pancreatic
Cancer Action Network Career Development Award (D.A. Tuveson), David Koch
Graduate Fellowship (K.P. Olive), and National Cancer Institute Mouse Models of
Human Cancer Consortium.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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The Differential Effects of Mutant p53 Alleles on Advanced
Murine Lung Cancer
Erica L. Jackson, Kenneth P. Olive, David A. Tuveson, et al.

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